Single Chelator–Minibody Theranostic Agents for 89Zr PET Imaging and 177Lu Radiopharmaceutical Therapy of PSMA-Expressing Prostate Cancer

Visual Abstract

The bone marrow toxicity observed for 177 Lu-DOTA-J591 is thought to arise from at least 2 potential sources.First, the slower clearance rate of antibody constructs from blood circulation is thought to innately contribute to increased toxicity (15).Second, antibody constructs cannot be heated to the temperatures necessary for complete incorporation of 177 Lu into the DOTA chelator (16,17), and yet their long circulation times necessitate even greater chelate stability than for rapidly excreted small-molecule agents.Incorporation of trivalent rare earth metal ions into DOTA proceeds slowly through 2 out-of-cage intermediates, necessitating high temperatures to form the in-cage complex (18).The directly radiolabeled 177 Lu-DOTA-antibody constructs may suffer from significant out-of-cage binding of 177 Lu, a condition for which discrimination via a convenient analytic technique does not exist to the best of our knowledge (19,20).
Here we investigated the effects of 2 potential improvements to 177 Lu-DOTA-J591.First, we made use of a minibody (80 kDa) derivative of J591 (IAB2MA; ImaginAb Inc.).The related 89 Zr-DFO-IAB2M minibody construct exhibited faster clearance rates and earlier peak signal-to-background ratios than did the analogous 89 Zr-DFO-J591 antibody construct (11,21).Compared with IgG antibodies, minibodies generally exhibit faster clearance and earlier tumor accumulation, and the Fc receptor-mediated interactions have been eliminated (22,23).Second, we evaluated a recently developed macrocyclic chelator, Lumi804 (L804; Lumiphore Inc.), which can be radiolabeled with 177 Lu (and 89 Zr) at room temperature and offers a stability advantage over the current state-of-the-art DOTA (and DFO) chelators.
The biodistribution, therapeutic efficacy, SPECT imaging, and dosimetry (using 2 methods: based on organ-level and voxel-level dose calculations) of 177 Lu-L804-IAB2MA and 177 Lu-DOTA-IAB2MA were compared in the PSMA-expressing (PC3-PIP) prostate tumor-bearing mouse model, along with the biodistribution and PET imaging of 89 Zr-L804-IAB2MA and 89 Zr-DFO-IAB2MA.In addition to the potential advantages for any one radionuclide, the L804 chelator binds both 177 Lu and 89 Zr exceptionally well, as demonstrated previously (24).In contrast, 89 Zr labeling of DOTA is challenging (25,26), and 177 Lu-labeled DFO lacks sufficient in vivo stability.The comparison of murine-model performance between L804 and DFO antibody constructs labeled with 89 Zr has been previously reported, and the advantage of L804 was found to be most apparent in decreased bone radioactivity at later time points (24).L804 has also been recently compared with DOTA and other chelators for 227 Th and 89 Zr labeled ofatumumab constructs (27).This report represents the first direct comparison of L804 and DOTA constructs with 177 Lu, using a well-validated cancer target and an improved biologic vector, with the aim of improving treatment and imaging of prostate cancer.

MATERIALS AND METHODS
Details on reagents (28), animal husbandry, minibody construct preparation, radiolabeling, Eu-L804-IAB2MA binding assays (24), radiolabeled internalization assays (29), and statistical analysis are included in the supplemental methods (supplemental materials are available at http://jnm.snmjournals.org).All animal experiments were conducted in compliance with the Institutional Animal Care and Use Committee at the University of Missouri.

Biodistribution of Radiolabeled Constructs
At 21 d after implantation of PC3-PIP prostate cancer cells, the mice were injected via tail vein with 89 Zr-L804-IAB2MA, 89 Zr-DFO-IAB2MA, 177 Lu-L804-IAB2MA, or 177 Lu-DOTA-IAB2MA and euthanized at time points from 4 to 96 h afterward, with blocking dose groups at 24 h (4 mice per group per time point).For the 89 Zr agents, each mouse received 100 mL of either 0.185 MBq (4, 24, and 48 h) or 0.37 MBq (72 h) of 89 Zr-L804-IAB2MA or 89 Zr-DFO-IAB2MA at a molar activity of 1.85 MBq/nmol.For the 177 Lu agents, each mouse received 100 mL of either 0.185 MBq (4 and 24 h) or 0.74 MBq (48, 72, and 96 h) of 177 Lu-L804-IAB2MA or 177 Lu-DOTA-IAB2MA at a molar activity of 7.4 MBq/nmol.Mice preloaded with unlabeled minibody (10 mg/kg) before the injection of the radiotracers were used as blocking control groups.Biodistribution of all radiotracers was also performed on PC3-FLU tumor-bearing mice at 24 h after injection (supplemental methods).At each time point, organs were collected and weighed.Tissue-associated radioactivity was determined in an automatic g-counter (1480 Wizard 399; PerkinElmer) and is expressed as percentage injected dose (%ID)/g.PET/CT and SPECT/CT Imaging PET/CT and SPECT/CT imaging studies (Albira Si; Bruker) were performed on PC3-PIP tumor-bearing mice at 21 d after implantation.The mice were injected via the tail vein with 100 mL of 89 Zr-L804-IAB2MA or 89 Zr-DFO-IAB2MA (5.55 MBq) at a molar activity of 9.25 MBq/nmol or with 177 Lu-L804-IAB2MA or 177 Lu-DOTA-IAB2MA (22.2 MBq) at a molar activity of 18.5 MBq/nmol (3 male mice per group).For PET imaging, mice injected with the 89 Zr agents were scanned at 4, 24, and 48 h after injection.For the SPECT/CT imaging, mice injected with the 177 Lu agents were scanned at 4, 24, 48, and 72 h after injection.Regions of interest were drawn using the CT scan, and the associated PET activities were calculated using Imalytics Preclinical 3.1 (Gremse-IT) (30) and expressed as %ID/g.

Targeted Radiopharmaceutical Therapy
At 9 d after implantation of PC3-PIP tumor cells (1 3 10 7 cells per mouse) in the right flank, the mice were randomized to provide an average tumor volume of approximately 6 mm 3 and an average body weight of approximately 24 g in each group (8 male mice).The mice received single therapeutic doses of 177 Lu-L804-IAB2MA or 177 Lu-DOTA-IAB2MA (14.8 and 22.2 MBq; 100 mL; 18.5 MBq/nmol).Non-tumor-bearing mice were included as a control group (blood control, 8 male mice).An additional control group (tumor-bearing mice) received unlabeled minibody (2.2 mg/kg).Tumor volumes were measured (length 3 width 3 height), and the mice were monitored twice weekly for weight loss.Blood was drawn by tail vein and was collected into ethylenediaminetetraacetic acid-coated tubes at 2 d before the treatment and every 2 wk thereafter for complete blood count analysis (VetScan HM5 hematology analyzer; Abaxis).Mice that were found in a moribund condition, had at least 20% body weight loss, or had a tumor burden of at least 1,500 mm 3 reached the endpoint and were removed from the study.At the time of euthanasia, all major organs were harvested and analyzed for signs of radiotoxicity.Data were plotted as a Kaplan-Meier graph of time when the mice were removed from the study.

Synthesis and Radiolabeling Chemistry
Chelator-to-minibody ratios of 2-2.5 were achieved for all conjugates (Supplemental Figs. 1 and 2).Quantitative radiolabeling of the L804-IAB2MA construct in ammonium acetate buffer with 89 Zr was achieved at ambient temperature in under 30 min at a molar activity of 9.25 MBq/nmol, which was comparable to the quantitative labeling of the DFO-IAB2MA construct under the same conditions in 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (.99% determined by size-exclusion high-performance liquid chromatography).Similarly, L804-IAB2MA was radiolabeled with 177 Lu in under 30 min at ambient temperature at a molar activity of 18.5 MBq/nmol in high radiochemical yields and purity (.99% by size-exclusion high-performance liquid chromatography).In contrast, DOTA-IAB2MA was radiolabeled with 177 Lu for 30 min at 37 C, and only about 90% radiochemical yield could be obtained, consistent with previous reports (33).The reaction mixture was purified using Centricon (MilliporeSigma) filtration to yield 177 Lu-DOTA-IAB2MA with purity of more than 99% and a purification yield of more than 85%.All 4 agents exhibited high stability (.95%) for 72 h at 37 C in mouse serum (Supplemental Fig. 3).

Binding Affinity of Eu-L804-IAB2MA
When europium(III) was used as a luminescent surrogate metal ion, the binding affinity of Eu-L804-IAB2MA was measured in PSMA-positive PC3-PIP and PSMA-negative PC3-FLU prostate cancer cells by time-resolved luminescence.The Eu-L804-IAB2MA conjugate possessed high binding affinity to PSMA in PC3-PIP cells (dissociation constant, 4.6 6 0.6 nM) (Supplemental Fig. 4).Saturation binding could not be determined for PC3-FLU cells, as total (nonspecific plus specific) and nonspecific binding were indistinguishable.

h 4 h 24 h 24 h 48 h 48 h Time after injection (h) 4 h 4 h 24 h 24 h 48 h 48 h Time after injection (h) 4 h 4 h 24 h 24 h 48 h 48 h Time after injection (h)
Supplemental Table 12).At 19 d, the white blood cell and lymphocyte counts were significantly lower in mice receiving 177 Lu-DOTA-IAB2MA than in mice receiving 177 Lu-L804-IAB2MA (P , 0.05).
One mouse treated with 177 Lu-DOTA-IAB2MA (22.2 MBq) exhibited weight loss and very low white blood cell and lymphocyte counts and eventually died at 58 d after treatment, although there was no palpable tumor at 30 d after treatment and onward (Supplemental Fig. 14).

DISCUSSION
Biodistribution studies comparing 89 Zr-DFO-IAB2MA with 89 Zr-L804-IAB2MA show statistically significant differences in tissue localization of the 2 agents at 72 h after injection (Fig. 1).A greater retention of 89 Zr-DFO-IAB2MA in off-target tissues (e.g., spleen, kidney, heart, lacrimal/salivary glands, and bone) is consistent with reticuloendothelial system uptake and may be in part attributable to loss of 89 Zr from DFO. Biodistribution studies comparing 177 Lu-DOTA-IAB2MA with 177 Lu-L804-IAB2MA also show significant differences in tissue localization at 96 h after injection (Fig. 2).Specifically, greater retention of 177 Lu-DOTA-IAB2MA in off-target tissues (e.g., spleen, kidney, heart, lacrimal/salivary glands, and bone) is consistent with reticuloendothelial system uptake and loss of the metal ion by the DOTA chelator.The kidney uptake of 177 Lu-DOTA-IAB2MA is similar to results reported for 177 Lu-CHX-A99-DTPA-A11, in a study that did not pursue that agent further because of excessive kidney localization (34).
PET/SPECT and PET/CT imaging studies and dosimetry analyses generally support the conclusions of the biodistribution data (Figs. 3 and 4), particularly the faster clearance of 177 Lu-L804-IAB2MA than of 177 Lu-DOTA-IAB2MA.Both the biodistribution and the imaging studies indicate (at varying levels of significance) that there is greater localization of 177 Lu-DOTA-IAB2MA and 89 Zr-DFO-IAB2MA in PC3-PIP tumors than of the respective radiolabeled L804 conjugates.However, in both cases, the increased tumor localization is offset by the increased retention in nontarget organs, and the absorbed doses by the kidney, liver, spleen, muscle, and salivary glands are also lower for 177 Lu-L804-IAB2MA (Table 1).At equivalent injected amounts of 177 Lu, agents that clear more rapidly are expected to result in lower levels of tumor localization.However, after normalization for absorbed tumor dose, tumor-to-kidney and tumor-to-spleen ratios favor 177 Lu-L804-IAB2MA.From the absorbed doses, it is clear that these minibody agents are less dependent on renal excretion than are analogous small- molecule agents, particularly for the L804 derivative (35).Absorbed doses to the PC3-PIP tumors (0.21-0.73 Gy/MBq) were similar to those reported for 177 Lu-PSMA-617 in C4-2 cells (0.0758 Gy/MBq) and LNCaP cells (0.594 Gy/MBq) (36,37).Estimates of kidney doses for 177 Lu-PSMA-617 vary from 0.07 to 0.55 Gy/MBq (38), and the uncertainty derives from the fact that it is more difficult to accurately quantify critical early time points.As salivary glands are one of the dose-limiting organs for smallmolecule agents (39), the avoidance of salivary gland localization by minibody-based radiotherapeutics could facilitate efficacious tumor doses at the expense of different dose-limiting organs (e.g., liver and bone marrow).
The efficacy of 177 Lu-DOTA-IAB2MA and 177 Lu-L804-IAB2MA after single-dose administration was compared in the PC3-PIP tumor regrowth model (Fig. 5).Although survival did not statistically differ between treatment groups, tumor growth data showed better growth control for 177 Lu-DOTA-IAB2MA than for 177 Lu-L804-IAB2MA, where 3 of 8 tumors regrew after treatment in the lower-dose group (14.8 MBq/mouse) and 1 tumor regrew in the higher-dose group (22.2 MBq/mouse).Tumor growth was effectively controlled in all other mice outside of the unlabeled minibody control group.When equivalent administered amounts of radioactivity were compared, the greater efficacy of 177 Lu-DOTA-IAB2MA was consistent with the increased tumor localization from the imaging and biodistribution studies.However, there were also indications that these doses of 177 Lu-DOTA-IAB2MA were less well tolerated, especially in the higher-dose (22.2 MBq) group, where 1 mouse of 8 failed to thrive after treatment.Complete blood count analysis showed a more pronounced depletion and slower recovery of leukocytes and lymphocytes after treatment with the DOTA agent (Fig. 6).Weight loss after treatment corroborated the hematology data, with mice recovering weight more quickly in the 177 Lu-L804-IAB2MA groups than in the equivalent 177 Lu-DOTA-IAB2MA treatment groups.
Taken together, we expect the maximum tolerated dose to be higher for 177 Lu-L804-IAB2MA than for 177 Lu-DOTA-IAB2MA, which may allow for equivalent or better tumor growth inhibition with less hematologic toxicity.If the slower clearance of 177 Lu-DOTA-IAB2MA is due to loss of the metal ion in vivo, this effect may be more pronounced in humans because of the faster clearance generally observed in mice.Future studies would benefit from examination of further dose escalation for 177 Lu-L804-IAB2MA and investigation in additional PSMA-positive tumor-bearing mouse models.

CONCLUSION
The macrocyclic chelator L804 was compared with the current gold standard chelators DOTA and DFO as PSMA-directed IAB2MA minibody constructs in preclinical biodistribution, imaging, dosimetry, and efficacy studies.Although the 89 Zr-and 177 Lu-L804-IAB2MA conjugates showed lower tumor uptake, they generally exhibited faster nontarget organ clearance than the corresponding 89 Zr-DFO or 177 Lu-DOTA agents, consistent with more stable radiometal chelation.The faster clearance was also consistent with reduced salivary gland localization and reduced hematopoietic toxicity.Continued investigation of the theranostic 89 Zr-and 177 Lu-L804-IAB2MA agents as alternatives to current PSMA-directed radiotheranostic agents is ongoing.
177Lu-L804-IAB2MA (14.8 or 22.2 MBq) and non-tumor-bearing mice, whereas mice receiving 177 Lu-DOTA-IAB2MA (14.8 or 22.2 MBq) had significantly lower weight than did non-tumor-bearing mice during the study.Complete blood count analysis indicated a transient decrease in the white blood cell and lymphocyte subsets at 5 d after treatment, which recovered within 19 d for the177 Lu-L804-IAB2MA-treated groups and by 33 for the 177 Lu-DOTA-IAB2MAtreated groups (Fig.6; Supplemental Fig.13;

DISCLOSURE
This research was funded by the NIH Small Business Innovation Research (SBIR) Program (grant 1R43CA265652).David Tatum and Darren Magda are employed by and own stock options in Lumiphore, Inc. Fang Jia and Alessandro Mascioni are employed by ImaginAb Inc. Carolyn Anderson is on the scientific advisory board of and has funding from Lumiphore, Inc.No other potential conflict of interest relevant to this article was reported.